Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

Engineering aligned musculoskeletal and cardiac tissues remains a key challenge due to the lack of mechanical cues in conventional bioinks. Here, we present a synergistic strategy combining fiber-reinforced collagen bioinks with a brush-assisted bioprinting (BAB) process to fabricate highly organized, functional tissue constructs. The bioink incorporates straight and coiled poly(ε-caprolactone) (PCL) microfibers to deliver spatially defined biophysical guidance, while the BAB process applies directional and homogeneous shear stress to align both fibers and cells during printing. This integrated approach enhances cytoskeletal organization and activates mechanosensitive pathways, including <i>YAP/TAZ</i> and <i>PIEZO1</i>, promoting myogenic differentiation. <i>In vitro</i>, printed bioconstructs using C2C12 and H9C2 exhibited improved cell alignment, gene expression, and structural maturation. In a murine volumetric muscle loss (VML) model, BAB-fabricated human adipose-stem cell (hASC)-laden constructs restored muscle mass and function more effectively than control, with the coiled fiber group showing the significantly meaningful levels of muscle regeneration, reduced fibrosis, and human cell integration. Based on these results, this work can demonstrate a new platform for fabricating anisotropic tissue constructs and offer significant potential for translational applications in regenerative medicine.